Publications by authors named "Denise M Monack"

92 Publications

A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature.

PLoS Pathog 2021 Mar 2;17(3):e1009345. Epub 2021 Mar 2.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1009345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954313PMC
March 2021

A Rapid Caspase-11 Response Induced by IFN Priming Is Independent of Guanylate Binding Proteins.

iScience 2020 Oct 29;23(10):101612. Epub 2020 Sep 29.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFN priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFN treatment. Indeed, IFN priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFN priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFN.
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http://dx.doi.org/10.1016/j.isci.2020.101612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566093PMC
October 2020

Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.

PLoS Pathog 2020 08 24;16(8):e1008763. Epub 2020 Aug 24.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.
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http://dx.doi.org/10.1371/journal.ppat.1008763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446830PMC
August 2020

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

mBio 2020 06 23;11(3). Epub 2020 Jun 23.

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents. Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.
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http://dx.doi.org/10.1128/mBio.01293-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315125PMC
June 2020

Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells.

Gastroenterology 2020 07 1;159(1):214-226.e1. Epub 2020 Apr 1.

Palo Alto Veterans Institute of Research, VA Palo Alto Health Care System, Palo Alto, California; Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University, Stanford, California; Department of Microbiology and Immunology, Stanford University, Stanford, California. Electronic address:

Background & Aims: Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids.

Methods: We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells.

Results: A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids.

Conclusions: We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.
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http://dx.doi.org/10.1053/j.gastro.2020.03.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569531PMC
July 2020

Editorial overview: The fortunate students, a tribute to the fortunate professor.

Curr Opin Microbiol 2020 04 12;54:iii-vi. Epub 2020 Mar 12.

Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, USA. Electronic address:

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http://dx.doi.org/10.1016/j.mib.2020.02.001DOI Listing
April 2020

Editorial: Protein Export and Secretion Among Bacterial Pathogens.

Front Cell Infect Microbiol 2019 22;9:473. Epub 2020 Jan 22.

Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, Aix-Marseille University, Marseille, France.

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http://dx.doi.org/10.3389/fcimb.2019.00473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987241PMC
August 2020

Salmonella-Driven Polarization of Granuloma Macrophages Antagonizes TNF-Mediated Pathogen Restriction during Persistent Infection.

Cell Host Microbe 2020 01 26;27(1):54-67.e5. Epub 2019 Dec 26.

Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA. Electronic address:

Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium (STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11bCD11cLy6C macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.
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http://dx.doi.org/10.1016/j.chom.2019.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065835PMC
January 2020

Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization.

Cell Host Microbe 2020 01 17;27(1):41-53.e6. Epub 2019 Dec 17.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK. Electronic address:

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.
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http://dx.doi.org/10.1016/j.chom.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953433PMC
January 2020

Host inflammasome defense mechanisms and bacterial pathogen evasion strategies.

Curr Opin Immunol 2019 10 4;60:63-70. Epub 2019 Jun 4.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Inflammasomes are a formidable armada of intracellular pattern recognition receptors. They recognize determinants of infection, such as foreign entities or danger signals within the host cell cytosol, rapidly executing innate immune defenses and initiating adaptive immune responses. Although inflammasomes are implicated in many diseases, they are especially critical in host protection against intracellular bacterial pathogens. Given this role, it is not surprising that many pathogens have evolved effective strategies to evade inflammasome activation. In this review, we will provide a brief summary of inflammasome activation during infection with the intent of highlighting recent advances in the field. Additionally, we will review known bacterial evasion strategies and countermeasures that impact pathogenesis.
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http://dx.doi.org/10.1016/j.coi.2019.05.001DOI Listing
October 2019

Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-κB Signaling Dynamics in Single Host Cells.

Cell Syst 2019 03 20;8(3):183-196.e4. Epub 2019 Mar 20.

Department of Bioengineering, Stanford, CA, USA; Allen Discovery Center for Systems Modeling of Infection, Stanford, CA, USA. Electronic address:

During an infection, immune cells must identify the specific level of threat posed by a given bacterial input in order to generate an appropriate response. Given that they use a general non-self-recognition system, known as Toll-like receptors (TLRs), to detect bacteria, it remains unclear how they transmit information about a particular threat. To determine whether host cells can use signaling dynamics to transmit contextual information about a bacterial stimulus, we use live-cell imaging to make simultaneous quantitative measurements of host MAPK and NF-κB signaling, two key pathways downstream of TLRs, and bacterial infection and load. This combined, single-cell approach reveals that NF-κB and MAPK signaling dynamics are sufficient to discriminate between (1) pathogen-associated molecular patterns (PAMPs) versus bacteria, (2) extracellular versus intracellular bacteria, (3) pathogenic versus non-pathogenic bacteria, and (4) the presence or absence of features indicating an active intracellular bacterial infection, such as replication and effector secretion.
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http://dx.doi.org/10.1016/j.cels.2019.02.008DOI Listing
March 2019

Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions.

Cell Rep 2019 02;26(9):2509-2520.e4

Department of Pediatrics, Division of Infectious Diseases, Stanford University, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA. Electronic address:

Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. However, accessing the apical enteroid surface is challenging because it is enclosed within the spheroid. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cell extrusion. Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. This model will enable further examination of IECs in health and disease.
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http://dx.doi.org/10.1016/j.celrep.2019.01.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391775PMC
February 2019

Western diet regulates immune status and the response to LPS-driven sepsis independent of diet-associated microbiome.

Proc Natl Acad Sci U S A 2019 02 11;116(9):3688-3694. Epub 2019 Feb 11.

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305;

Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.
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http://dx.doi.org/10.1073/pnas.1814273116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397595PMC
February 2019

Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.

PLoS Biol 2019 01 15;17(1):e3000059. Epub 2019 Jan 15.

Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.
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http://dx.doi.org/10.1371/journal.pbio.3000059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333337PMC
January 2019

The oxidized phospholipid oxPAPC protects from septic shock by targeting the non-canonical inflammasome in macrophages.

Nat Commun 2018 03 8;9(1):996. Epub 2018 Mar 8.

Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, 60611, USA.

Lipopolysaccharide (LPS) of Gram-negative bacteria can elicit a strong immune response. Although extracellular LPS is sensed by TLR4 at the cell surface and triggers a transcriptional response, cytosolic LPS binds and activates non-canonical inflammasome caspases, resulting in pyroptotic cell death, as well as canonical NLRP3 inflammasome-dependent cytokine release. Contrary to the highly regulated multiprotein platform required for caspase-1 activation in the canonical inflammasomes, the non-canonical mouse caspase-11 and the orthologous human caspase-4 function simultaneously as innate sensors and effectors, and their regulation is unclear. Here we show that the oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) inhibits the non-canonical inflammasome in macrophages, but not in dendritic cells. Aside from a TLR4 antagonistic role, oxPAPC binds directly to caspase-4 and caspase-11, competes with LPS binding, and consequently inhibits LPS-induced pyroptosis, IL-1β release and septic shock. Therefore, oxPAPC and its derivatives might provide a basis for therapies that target non-canonical inflammasomes during Gram-negative bacterial sepsis.
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http://dx.doi.org/10.1038/s41467-018-03409-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843631PMC
March 2018

LysMD3 is a type II membrane protein without an role in the response to a range of pathogens.

J Biol Chem 2018 04 1;293(16):6022-6038. Epub 2018 Mar 1.

From the Departments of Pathology and Immunology and

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule .
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http://dx.doi.org/10.1074/jbc.RA117.001246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912457PMC
April 2018

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library.

Bio Protoc 2017 May;7(10)

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA.

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier ., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.
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http://dx.doi.org/10.21769/BioProtoc.2320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580966PMC
May 2017

T6SS: The bacterial "fight club" in the host gut.

PLoS Pathog 2017 06 8;13(6):e1006325. Epub 2017 Jun 8.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1006325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464660PMC
June 2017

Cell-Intrinsic Defense at the Epithelial Border Wall: Salmonella Pays the Price.

Immunity 2017 04;46(4):522-524

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

Within the gut, Salmonella-infected enterocytes are expelled into the lumen, limiting pathogen replication. In this issue of Immunity, Rauch et al. (2017) expand our understanding of this cell-intrinsic response by characterizing the genetic determinants that control the expulsion and death of epithelial cells.
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http://dx.doi.org/10.1016/j.immuni.2017.03.021DOI Listing
April 2017

Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity.

J Exp Med 2016 10 3;213(11):2365-2382. Epub 2016 Oct 3.

Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305

Cell death and release of proinflammatory mediators contribute to mortality during sepsis. Specifically, caspase-11-dependent cell death contributes to pathology and decreases in survival time in sepsis models. Priming of the host cell, through TLR4 and interferon receptors, induces caspase-11 expression, and cytosolic LPS directly stimulates caspase-11 activation, promoting the release of proinflammatory cytokines through pyroptosis and caspase-1 activation. Using a CRISPR-Cas9-mediated genome-wide screen, we identified novel mediators of caspase-11-dependent cell death. We found a complement-related peptidase, carboxypeptidase B1 (Cpb1), to be required for caspase-11 gene expression and subsequent caspase-11-dependent cell death. Cpb1 modifies a cleavage product of C3, which binds to and activates C3aR, and then modulates innate immune signaling. We find the Cpb1-C3-C3aR pathway induces caspase-11 expression through amplification of MAPK activity downstream of TLR4 and Ifnar activation, and mediates severity of LPS-induced sepsis (endotoxemia) and disease outcome in mice. We show C3aR is required for up-regulation of caspase-11 orthologues, caspase-4 and -5, in primary human macrophages during inflammation and that c3aR1 and caspase-5 transcripts are highly expressed in patients with severe sepsis; thus, suggesting that these pathways are important in human sepsis. Our results highlight a novel role for complement and the Cpb1-C3-C3aR pathway in proinflammatory signaling, caspase-11 cell death, and sepsis severity.
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http://dx.doi.org/10.1084/jem.20160027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068231PMC
October 2016

Editorial: Bacterial Exotoxins: How Bacteria Fight the Immune System.

Front Immunol 2016 2;7:300. Epub 2016 Aug 2.

Zentrum für Infektiologie, Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Heidelberg , Heidelberg , Germany.

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http://dx.doi.org/10.3389/fimmu.2016.00300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970444PMC
August 2016

Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut.

Proc Natl Acad Sci U S A 2016 08 8;113(34):E5044-51. Epub 2016 Aug 8.

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305;

The mammalian gastrointestinal tract is colonized by a high-density polymicrobial community where bacteria compete for niches and resources. One key competition strategy includes cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), a multiprotein needle-like apparatus that injects effector proteins into prokaryotic and/or eukaryotic target cells. However, the contribution of T6SS antibacterial activity during pathogen invasion of the gut has not been demonstrated. We report that successful establishment in the gut by the enteropathogenic bacterium Salmonella enterica serovar Typhimurium requires a T6SS encoded within Salmonella pathogenicity island-6 (SPI-6). In an in vitro setting, we demonstrate that bile salts increase SPI-6 antibacterial activity and that S Typhimurium kills commensal bacteria in a T6SS-dependent manner. Furthermore, we provide evidence that one of the two T6SS nanotube subunits, Hcp1, is required for killing Klebsiella oxytoca in vitro and that this activity is mediated by the specific interaction of Hcp1 with the antibacterial amidase Tae4. Finally, we show that K. oxytoca is killed in the host gut in an Hcp1-dependent manner and that the T6SS antibacterial activity is essential for Salmonella to establish infection within the host gut. Our findings provide an example of pathogen T6SS-dependent killing of commensal bacteria as a mechanism to successfully colonize the host gut.
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http://dx.doi.org/10.1073/pnas.1608858113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003274PMC
August 2016

Microbiology: The dark side of antibiotics.

Nature 2016 06 15;534(7609):624-5. Epub 2016 Jun 15.

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http://dx.doi.org/10.1038/nature18449DOI Listing
June 2016

IMMUNOLOGY. A lipid arsenal to control inflammation.

Science 2016 Jun 2;352(6290):1173-4. Epub 2016 Jun 2.

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94306, USA.

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http://dx.doi.org/10.1126/science.aag0366DOI Listing
June 2016

Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death.

Elife 2016 Mar 24;5:e13663. Epub 2016 Mar 24.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, United States.

When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.
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http://dx.doi.org/10.7554/eLife.13663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846378PMC
March 2016

Variation in Taxonomic Composition of the Fecal Microbiota in an Inbred Mouse Strain across Individuals and Time.

PLoS One 2015 13;10(11):e0142825. Epub 2015 Nov 13.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Genetics, diet, and other environmental exposures are thought to be major factors in the development and composition of the intestinal microbiota of animals. However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood. We studied a population of inbred, female mice fed the same diet and housed under the same conditions. We collected fecal samples from 46 individual mice over two weeks, sampling four of these mice for periods as long as 236 days for a total of 190 samples, and determined the phylogenetic composition of their microbial communities after analyzing 1,849,990 high-quality pyrosequencing reads of the 16S rRNA gene V3 region. Even under these controlled conditions, we found significant inter-individual variation in community composition, as well as variation within an individual over time, including increases in alpha diversity during the first 2 months of co-habitation. Some variation was explained by mouse membership in different cage and vendor shipment groups. The differences among individual mice from the same shipment group and cage were still significant. Overall, we found that 23% of the variation in intestinal microbiota composition was explained by changes within the fecal microbiota of a mouse over time, 12% was explained by persistent differences among individual mice, 14% by cage, and 18% by shipment group. Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time. These findings have implications for the design and interpretation of experiments involving laboratory animals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142825PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643986PMC
June 2016

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin.

J Immunol 2015 Aug 24;195(3):815-9. Epub 2015 Jun 24.

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6.
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http://dx.doi.org/10.4049/jimmunol.1403100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505955PMC
August 2015

IMMUNOLOGY. Microbial metabolite triggers antimicrobial defense.

Science 2015 Jun;348(6240):1207-8

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305, USA.

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http://dx.doi.org/10.1126/science.aac5835DOI Listing
June 2015

Bacterial recognition pathways that lead to inflammasome activation.

Immunol Rev 2015 May;265(1):112-29

Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA.

Inflammasomes are multi-protein signaling platforms that upon activation trigger the maturation of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and IL-18, and cell death. Inflammasome sensors detect microbial and host-derived molecules. Here, we review the mechanisms of inflammasome activation triggered by bacterial infection, primarily focusing on two model intracellular bacterial pathogens, Francisella novicida and Salmonella typhimurium. We discuss the complex relationship between bacterial recognition through direct and indirect detection by inflammasome sensors. We highlight regulation mechanisms that potentiate or limit inflammasome activation. We discuss the importance of caspase-1 and caspase-11 in host defense, and we examine the downstream consequences of inflammasome activation within the context of bacterial infections.
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http://dx.doi.org/10.1111/imr.12289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437016PMC
May 2015